We have previously shown that acetylcholine (ACh) induces oscillations in Ca++ and Ca++-activated Cl- currents (Clca) in tracheal myocytes. These oscillations are initiated by Ca++ release from inositol 1,4,5-trisphosphate-sensitive Ca++ stores and maintained by Ca++ influx, in part, through voltage-operated Ca++ channels. In the current study whole-cell Clca was measured in isolated tracheal smooth muscle cells as an index of changes in intracellular Ca++ concentration. We demonstrate that ACh-sensitive Ca++ stores and caffeine-sensitive Ca++ stores and caffeine-sensitive Ca++ stores are functionally linked but are refilled through distinct pathways. Two pathways responsible for replenishing ACh-sensitive Ca++ stores were identified. Ca++ influx through verapamil-sensitive voltage-operated Ca++ channels and Ca++ uptake through cyclopiazonic acid-sensitive Ca++ pumps accounted for 80% of the response. The other 20% of the response was both cyclopiazonic acid- and verapamil-insensitive. In contrast, the refilling of caffeine-sensitive Ca++ stores was not inhibited by 10 microM cyclopiazonic acid or 10 microM verapamil, but was dependent on extracellular Ca++ concentration. However, 0.2 microM thapsigargin, another more potent Ca++ pump inhibitor, completely and irreversibly eliminated ACh-induced transient Clca, whereas it reduced caffeine-induced Clca by 57%. The differences in refilling mechanisms and the functional overlap of ACh- and caffeine-sensitive Ca++ pools suggest that multiple interactive Ca++ stores play an important role in the generation of Ca++ signals in airway smooth muscle cells.